Mean Myeloperoxidase Research Articles

Myeloperoxidase, Subclinical Atherosclerosis, and Cardiovascular Disease Events

We evaluated whether myeloperoxidase (MPO) predicts future cardiovascular disease (CVD) events in asymptomatic adults and whether subclinical atherosclerosis may affect this relation. Myeloperoxidase is a leukocyte-derived enzyme-generating reactive oxidant species that has been shown to predict risk of CVD in selected populations. We studied 1,302 asymptomatic adults (mean age 59 years, 47% women) without known CVD who were followed for 3.8 years. We measured MPO by the use of immunoassay. Coronary artery calcium (CAC), a measure of subclinical atherosclerosis, was measured by computed tomography with the Agatston score categorized as none/minimal (0 to 9), mild (10 to 99), and moderate/significant (> or = 100). Cox regression, adjusted for age, sex, and other risk factors, examined the relation of CAC and/or MPO with incident CVD events. Persons with MPO levels at or above compared with below the median (257 pM) were more likely (p < 0.05 to p < 0.001) to be women, have a higher body mass index, greater low-density lipoprotein cholesterol, greater systolic and diastolic blood pressure, and lower high-density lipoprotein cholesterol. Mean MPO levels increased according to CAC categories (p trend = 0.02). Incident CVD events were more likely in those at or above versus below the median MPO level (4.6% vs. 2.3%, p = 0.02), even after adjustment for age, sex, CAC, and risk factors (hazard ratio [HR]: 1.9, 95% confidence interval: 1.0 to 3.6, p = 0.04). Combining CAC and MPO categories, CVD incidence ranged from 0.6% in those with a CAC score of 0 to 9 to 7.1% (adjusted HR: 9.2, p < 0.001) in those with CAC scores of > or = 100 and MPO below the median and 14.0% (adjusted HR: 19.5, p < 0.0001) in those with CAC scores of > or = 100 and MPO at or above the median. Our study suggests persons with both increased levels of both MPO and CAC are at an increased risk of CVD events. Imaging of subclinical atherosclerosis combined with assessment of biomarkers of plaque vulnerability may help improve CVD risk stratification.

Association between myeloperoxidase polymorphisms and its plasma levels with severity of coronary artery disease

Objectives Myeloperoxidase (MPO) polymorphism −463 has been related to higher cardiovascular risk. This study was conducted to test whether the MPO promoter polymorphism −463A/G and MPO plasma levels are associated with coronary artery disease (CAD) severity. Design and methods Patients submitted to elective coronariography were enrolled, CAD severity was assessed and blood samples collected to identify the MPO polymorphism and its plasma levels. Results Genotypes were determined in 118 patients. Among these patients, 12 (10%) were homozygous for AA, 69 (58%) for GG and 37 (32%) were heterozygous. Mean MPO plasma levels were 8.6 ± 4.7 ng/mL for AA, 8.6 ± 7.0 ng/mL for AG and 9.4 ± 5.6 ng/mL for GG genotypes. The CAD severity was not associated with MPO genotypes ( p = 0.43), however, patients with higher CAD score presented higher MPO levels ( p = 0.02). Conclusion We found no association between MPO polymorphism and CAD severity, although a relation was observed for MPO plasma levels and extension of CAD.

Myeloperoxidase to Risk Stratify Emergency Department Patients with Chest Pain

Previous studies suggest that serum myeloperoxidase (MPO) is a potentially useful biomarker to risk stratify troponin-negative patients with suspected myocardial ischemia. We hypothesized that the relationship between initial serum MPO levels would correlate with 30-day adverse cardiac outcomes for low risk emergency department (ED) patients with suspected myocardial ischemia. This prospective cohort study enrolled ED patients with chest pain or suspected myocardial ischemia, non-diagnostic ECG, and initially negative cardiac troponin I. We defined 30-day adverse cardiac events as death, myocardial infarction, or coronary revascularization. We calculated summary statistics, standard deviation (SD), odds ratios (OR), 95% confidence intervals (CI), and receiver operating characteristics (ROC). We enrolled 159 patients who had a mean age of 55 ± 13, were 56% female, of whom 5.2% suffered at least one adverse cardiac event. MPO test characteristics were poor, with an ROC area of only 0.47 (CI 0.23-0.71). MPO levels were not associated with adverse events (OR 0.99, CI 0.98-1.01, p=0.62). The optimal ROC cutpoint to predict adverse cardiac events had poor sensitivity and specificity (57% and 52%, respectively). Mean MPO concentrations in the event group did not differ from the non-event group. In this limited cohort of low risk ED patients with chest pain, we were unable to demonstrate utility of MPO for risk stratification. If confirmed in larger studies, these findings may call into question the routine use of MPO for low-risk chest pain.

The effects of montelukast on random pattern skin flap survival: An experimental study in rats

Background: A variety of methods to improve skin flap survival, including the use of pharmacologic agents, have been intensively investigated. Decreasing neutrophil-mediated inflammation and tissue injury has been reported to be effective in improving flap survival. Montelukast is a selective reversible cysteinyl leukotriene receptor-1 antagonist that has been found to have protective effects against renal ischemia reperfusion injury and burn-induced oxidative injury of the skin in rats. However, its effects on skin flap survival have not been previously reported. Objective: The aim of this study was to investigate the effects of montelukast on neutrophil-mediated random pattern skin flap survival. Methods: Male Sprague-Dawley rats weighing 230 to 250 g were randomly divided into 2 groups—the montelukast-treated group and the control group. Caudally based rectangular random pattern skin flaps 3 × 9 cm were elevated on the backs of the rats. The flaps were sutured into their original places. In the montelukast group, 1 mL of solution containing 10 mg/kg montelukast was administered intraperitoneally (IP) 30 minutes before surgery and then daily for 6 days. In the control group, 1 mL of saline was administered IP 30 minutes before surgery and then daily for 6 days. To observe the effects of montelukast, myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration, was measured from extracted skin tissue 12 hours after flap elevation. Flap viability was evaluated 7 days after surgery by measuring necrotic flap area and total flap area. Results: Sixteen rats (mean [SD] weight, 240.6 [6.6] g) were equally divided between the 2 groups. All rats survived throughout the study period. Mean (SD) MPO activity in flap tissue was significantly lower in the montelukast group than in the control group (14.57 [2.33] vs 21.28 [4.86] U/g protein; P = 0.005). The percentage of necrotic flap area was significantly lower in the montelukast group than in the control group (17.17 [7.95] vs 37.51 [10.72]; P = 0.001). Conclusion: This small, experimental, in vivo animal study found that montelukast was associated with both lower MPO activity and a lower percentage of necrotic random pattern skin flap area. Future studies are needed to clarify these findings.

Elevation of Myeloperoxidase in Conjunction With Cardiac-Specific Markers After Marathon Running

Cardiac-related death has been reported following strenuous exercise, and biochemical markers predicting adverse outcomes would be useful. Despite the fact the myeloperoxidase (MPO) release may precede myocardial injury and identify at-risk patients earlier than traditional markers, information on the effects of marathon running on MPO levels is lacking. We measured MPO in conjunction with the creatine kinase MB fraction (CK-MB), myoglobin, troponin T (TnT), and N-terminal B-type natriuretic peptide (NT-proBNP) in 24 athletes before and after a marathon race. Of the 24 athletes, 22 (92%) had an increased MPO level, and the mean MPO level increased from 281.44 pmol/L to 785.21 pmol/L (P < .0001). Results for 14 (58%) of the athletes reached or exceeded the manufacturer’s recommended clinical threshold. The increases in CK-MB, myoglobin, TnT, and NT-proBNP also reached statistical significance. Although the elevation in MPO most likely represents a systemic inflammatory response, the concurrent elevations in TnT and NT-proBNP suggest that myocardial injury cannot be excluded.

Elevation of Myeloperoxidase in Conjunction With Cardiac-Specific Markers After Marathon Running

Cardiac-related death has been reported following strenuous exercise, and biochemical markers predicting adverse outcomes would be useful. Despite the fact the myeloperoxidase (MPO) release may precede myocardial injury and identify at-risk patients earlier than traditional markers, information on the effects of marathon running on MPO levels is lacking. We measured MPO in conjunction with the creatine kinase MB fraction (CK-MB), myoglobin, troponin T (TnT), and N-terminal B-type natriuretic peptide (NT-proBNP) in 24 athletes before and after a marathon race. Of the 24 athletes, 22 (92%) had an increased MPO level, and the mean MPO level increased from 281.44 pmol/L to 785.21 pmol/L (P < .0001). Results for 14 (58%) of the athletes reached or exceeded the manufacturer's recommended clinical threshold. The increases in CK-MB, myoglobin, TnT, and NT-proBNP also reached statistical significance. Although the elevation in MPO most likely represents a systemic inflammatory response, the concurrent elevations in TnT and NT-proBNP suggest that myocardial injury cannot be excluded.

THE TRPV1 RECEPTOR ANTAGONIST JYL-1421 REDUCES COLONIC INFLAMMATION AND PREVENTS VISCERAL HYPERALGESIA IN A RATS MODEL OF EXPERIMENTAL COLITIS

Background: Visceral hyperalgesia results from transient colonic inflammation. The transient receptor potential vanilloid 1 (TRPV1) receptor may play an important role in visceral nociception and neurogenic inflammation. We aimed to examine the effects of the TRPV1 receptor antagonist (RA) JYL-1421 on visceral hypersensitivity and inflammation in a rat model of colitis. Methods: Acute inflammation was induced in male Sprague-Dawley rats by single administration of TNBS (trinitro benzenesulfonic acid) in the distal colon. Rats were randomized to receive either JYL-1421 (10 μmol) or saline. JYL-1421 was given intravenously 15 minutes prior to administration of TNBS followed by daily doses for 7 days. Control rats received i.v. saline on the same schedule. In all rats, a baseline visceromotor response (VMR) to graded colorectal distension (CRD) (10-80 mmHg) was recorded prior to TNBS. Seven days post TNBS, the VMR was repeated in both groups to assess alterations in visceral sensitivity. To assess the severity of inflammation, sections of the distal colon were examined with H&E staining and for myeloperoxidase (MPO) activity. Results: In the control group, intracolonic TNBS produced a significant increase in the VMR to CRD at distension pressures >10 mmHg (n = 18, P = 0.01). However, rats that received JYL-1421 did not develop hyperalgesia and failed to demonstrate any alteration in the VMR when compared to baseline (n = 16, P > 0.05). The VMR of control rats was significantly higher than the treated group at distension pressures >10 mmHg (P < 0.02). In control rats (n = 4), TNBS resulted in a marked increase in submucosal neutrophilic infiltrates that was markedly lower in the JYL-1421 treated group (n = 4). The mean MPO activity was significantly lower in the JYL-1421 treated group (7.98 ± 3.0, n = 6) when compared to controls (33.47 ± 8.6, n = 5) (P = 0.01). Conclusions: Intracolonic TNBS results in colonic inflammation and visceral hyperalgesia that can be attenuated by systemic administration of the TRPV1 RA, JYL-1422.

The Protective Effect of α-Tocopherol and GdCl3 Against Hepatic Ischemia/Reperfusion Injury

To evaluate the combined effect of alpha-tocopherol and gadolinium chloride (GdCl3) in reducing lipid peroxidation after severe hepatic ischemia/reperfusion (IR) injury. Sixty male Wistar rats, 200-250 g, were randomly divided into six equal groups. There were two sham operation (SHAM) groups, two untreated IR groups, and two IR groups treated with GdCl3 and alpha-tocopherol (IRGT). After 60 min of total hepatic ischemia and 120 min reperfusion, one of each group was killed, liver samples were taken for malondialdehyde (MDA) and myeloperoxidase (MPO) analysis and light microscopy examination, and blood samples were analyzed for aspartate (AST) and alanine (ALT) transaminase, lactate dehydrogenase (LDH), and alpha-tocopherol content. The remaining groups were monitored for survival rate determination. The mean MDA and MPO values in the SHAM, IR, and IRGT groups, respectively, were 1.117, 1.476, and 0.978 nmol/g wet tissue and 1.49, 6.26, and 1.78 (U/g). The mean alpha-tocopherol values in the SHAM, IR, and IRGT groups, respectively, were 10.4, 1.9, and 12 micromol/l. The mean serum AST, ALT, and LDH values were significantly higher in the IR group than in the SHAM group (P < 0.001), and significantly lower in the IRGT group than in the IR group (P < 0.001). Light microscopy examination revealed more severe congestion and vacuolization in the IR group than in the SHAM group, and minimal congestion and vacuolization in the IRGT group. Survival was significantly higher in the IRGT group than in the IR group. The administration of GdCl3 and alpha-tocopherol is likely to protect the liver against lipid peroxidation by suppressing Kupffer cell and polymorphonuclear leukocyte activation and enhancing endogenous antioxidant activity.

Attenuation of intestinal ischemia/reperfusion induced liver and lung injury by intraperitoneal administration of (−)-Epigallocatechin-3-gallate

The aim of this study was to evaluate the effect of ( − )-epigallocatechin-3-gallate (EGCG), a natural antioxidant, on liver and lungs after warm intestinal ischemia/reperfusion (I/R). Thirty male Wistar rats were equally divided into a sham-operation group, an intestinal I/R group and an intestinal I/R group pretreated with EGCG intraperitoneally. Intestinal ischemia was induced by occlusion of the superior mesenteric artery for 60 min followed by reperfusion for 120 min. Immediately after reperfusion, liver, lung and blood samples were collected and analyzed. Results showed that intestinal I/R increased the levels of aspartate (AST) and alanine (ALT) transaminase in serum to 987 and 752 IU/l, respectively. Malondialdehyde (MDA) increased in liver to 1.524 nmol/g in the group subjected to intestinal I/R compared to 0.995 nmol/g in the sham operation group. MDA was also increased in lungs to 1.581 nmol/g compared to 0.896 nmol/g in the sham operation group. Myeloperoxidase (MPO) increased in liver, after intestinal I/R, to 5.16 U/g compared to 1.59 U/g in the sham operation group. MPO was also increased in lungs to 3.89 U/g compared to 1.65 U/g in the sham operation group. Pretreatment with EGCG decreased serum levels of AST and ALT to 236 and 178 IU/l, respectively. It also decreased mean MDA levels in liver and lungs to 1.061 and 1.008 nmol/g, respectively, and mean MPO levels in liver and lungs to 1.88 and 1.71 U/g, respectively. Light microscopy and transmission electron microscopy examinations showed significant alteration in liver and lungs and protection of liver and lung parenchyma in the animals treated with EGCG.

The tripeptide phenylalanine-( d) glutamate-( d) glycine modulates leukocyte infiltration and oxidative damage in rat injured spinal cord

The tripeptide, phenylalanine-glutamate-glycine (FEG) and its d-isomeric form phenylalanine-( d) glutamate-( d) glycine (feG), derived from submandibular gland peptide-T, significantly reduce the allergic inflammatory response and leukocyte trafficking and neutrophil migration into intestine, heart and lungs. Due to these actions, we hypothesized that feG would attenuate the early inflammatory response to spinal cord injury, reduce free radical production and improve neurological outcomes, like other leukocyte-limiting strategies we have used previously. We tested this using a clip compression model of spinal cord injury in rats. Following spinal cord injury at the 4th thoracic cord segment, we quantified leukocyte infiltration, free radical formation and oxidative damage at the lesion site after feG or control peptide phenylalanine-( d) aspartate-( d) glycine treatment. In rats treated with feG at 2 and 12 h, or 6 and 12 h after spinal cord injury, mean myeloperoxidase activity and ED-1 expression were significantly lower (∼40%) than in controls at 24 h. Free radical formation generated in injured spinal cord was detected using 2′,7′-dichlorofluorescin-diacetate as a fluorescent probe. Free radical production in the injured cord increased significantly after spinal cord injury and feG treatment significantly reduced this free radical production. Oxidative enzymes, lipid peroxidation and cell death were also significantly (∼40%), gp91 (∼30%), thiobarbituric acid reactive substance levels (∼35%), 4-hydroxynonenal-bound protein (∼35%) and caspase-3 (∼32%). Early administration of feG decreases infiltration of inflammatory cells into the injured spinal cord and intraspinal free radical formation, thereby reducing oxidative damage and secondary cell death after spinal cord injury.

Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.

Endothelium-derived microparticles induce acute lung injury

Abstract Introduction: Elevated plasma levels of endothelium-derived microparticles (EMPs) have been detected in human disease states including sepsis. EMPs (0.1–4 microns) consist of a lipid bilayer with normal and abnormal endothelial cell surface proteins. Previous work has demonstrated EMP dependent endothelial dysfunction. We hypothesize that EMPs may contribute to pulmonary capillary leak and inflammation in acute lung injury (ALI). Methods: EMPs were generated by stimulating human umbilical vein endothelial cells (P4) with plasminogen activation inhibitor-1. Supernatant was collected, concentrated, and the presence of EMPs confirmed using flow cytometric analysis. Twenty-four Brown Norway rats were treated for six hours with intraperitoneal injection of normal saline, lipopolysaccharide (4 mg/kg), or intravenous injection of EMPs to attain plasma levels of 1 million/mL. As a measure of permeability, four rats per group were injected with I125 labeled albumin thirty minutes prior to sacrifice and I125 blood/lung ratios determined. Myeloperoxidase activity of homogenized lungs from each group was measured. Results: Normal saline, LPS, and EMP (n = 4/grp) mean lung permeability ratios and standard deviations were 0.0693+/−0.080, 0.166+/−0.037(p = 0.01), and 0.336+/−0.123(p = 0.03) respectively. Similarly, mean myeloperoxidase activity for the groups (n = 4/grp) were 0.071+/−0.017, 0.109+/−0.020(p = 0.01), and 0.137+/−0.039(p = 0.02) respectively. Preliminary data from PMN depletion studies suggest PMN dependence for EMP induced ALI. Conclusions: EMPs induce both pulmonary capillary leak and inflammatory response in our model of ALI. The plasma levels of EMPs in these experiments approximate those in human patients, although the role of EMPs in human ALI is unknown.

The Activity of Milk Leukocytes in Response to a Water-soluble Fraction of Mycobacterium phlei in Bovine Subclinical Mastitis

The effect of a water-soluble fraction (WSF) of a non-pathogenic strain of Mycobacterium phlei was studied in bovine subclinical mastitis (SCM) by measuring the myeloperoxidase and acid phosphatase enzyme levels in the milk leukocytes. Forty-five cows were divided into three equal groups. Group I, consisting of 15 healthy cows, served as the control, whereas groups II and III each contained 15 cows with subclinical mastitis on the basis of a positive reaction in the California mastitis test (CMT). The cows in group II received 100 microg of WSF in 5 ml sterile phosphate-buffered saline, pH 7.4 (PBS) once only, while those in group III received 5 ml sterile PBS daily for 7 days, both treatments being given by the intramammary route. Observations were made up to 30 days after treatment (AT). The CMT of the healthy milk was negative (0), whereas it ranged between 1 and 2 points in SCM. The somatic cell count (SCC) increased significantly (p < 0.05) on day 3, then fell steeply from day 7 up to day 30 AT in the cows in group II. A steady decrease in the total bacterial count (TBC) was observed in the group treated with WSF but the bacterial counts remained high in the groups treated with PBS. The mean acid phosphatase level was enhanced by 119% on day 3 AT in group II but only by 18.7% in the cows in group III. The mean myeloperoxidase level was enhanced by 100% in the cows in group II but only by 18% in those in group III on day 3 AT. This significant reduction in the bacterial load in infected cows caused by intramammary infusion of WSF may be due to activation of the microbicidal activity of the neutrophils, but this requires confirmation.

The impact of atopy on neutrophil activity in middle ear effusion from children and adults with chronic otitis media.

To identify the relationship of neutrophil activity to allergy as reflected by the level of myeloperoxidase (MPO) in ears of atopic patients with chronic otitis media with effusion (OME) by objective testing. Evidence of neutrophils was measured in the effusion of atopic patients with chronic OME. Atopy was determined by intradermal and/or in vitro testing of allergic reaction to 10 inhalants, 2 molds, and 5 foods. Effusion MPO was measured prospectively in 138 ears from 106 consecutive patients with chronic OME. A total of 86 (81%) of 106 patients with OME tested atopic by in vitro or in vivo testing. Excluding 36 ears with purulence, the mean MPO level was 3132 microg/L in 84 atopic vs 142 microg/L in 18 nonatopic ears (P<.001). A total of 78 (90%) of 87 patients with OME were atopic. The surprising finding of marked elevation of effusion MPO in atopic patients but very low levels in nonatopic patients (P < .001) suggests that atopy may contribute to elevated levels of neutrophil activity in OME. An atopic patient may respond differently from a nonatopic one to the microbial or viral products of acute inflammation owing to the presence of primed inflammatory cells. This study provides confirmation on a cellular level that neutrophils are an integral part of the inflammatory process in OME to a disproportionate degree among atopic patients.

Octreotide inhibits myeloperoxidase activity in rat uterus

Several studies have demonstrated that synthetic somatostatin-analog octreotide could affect the functions of the hypothalamus, anterior pituitary gland and ovary. To date, very few studies on the effect of octreotide on uterine function have been reported. We investigated the effects of octreotide on neutrophil functions in rat uterus. Twenty Sprague-Dawley rats were subjected to a standardized uterine horn excision. To evaluate the effect of octreotide, the rats were assigned randomly to treatment (octreotide 20 microgr/kg, SC) and control groups. After two weeks relaparotomy was performed and contralateral uterine horns were excised in the same standardized manner. The excised segment was washed in cold saline solution and was frozen in liquid nitrogen immediately and homogenized. Myeloperoxidase (MPO) and elastase concentrations were determined in homogenates of uterine horns. The data were analyzed by Mann-Whitney U test. In octreotide-treated rats the MPO activity was significantly reduced after 14 days (14.79+/-2.08 versus 6.62+/-1.72 U/mg protein; p=0.012). There was also a significant difference in mean MPO levels between the two groups at the end of the experiment (6.62+/- 1.72 versus 18.56+/-2.49 U/mg protein; p=0.001). The octreotide administration did not affect elastase levels significantly. No differences were found in mean elastase levels in both groups after the treatment. This study demonstrates that octreotide has a significant effect on the neutrophil function in terms of MPO activity in rat uterus. Further studies are needed to clarify the clinical relevance of the inhibitory effect of octreotide in the uterus and the possibility of its applications.

Selective cytotoxicity of 3-amino-L-tyrosine correlates with peroxidase activity.

In the presence of 3-amino-L-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10(6) cells versus 26.1 and 5.0 ng/10(6) cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and > 70% reduction in cell viability after 4 d of culture in the presence of 100 microg 3-AT per ml. Higher concentrations of 3-AT (up to 400 microg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1-3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 microg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 microg 3-AT per ml, but concentrations less than 400 microg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 microg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 microg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated "aminomelanin" formation is hypothesized.

Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125 I by a technique of self-labeling in the presence of 10 −4 M hydrogen peroxide. The RIA was performed by incubation of 100 μl diluted antiserum, 100 μl labeled MPO (±30 000 cpm) and 100 μl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody–antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 ± 19.4 ng/ml in EDTA anticoagulated plasma ( n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persitent abnormal MPO concentrations were measured (until 740 ng/ml).

Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation after hemorrhagic shock.

Hypertonic saline (HS) resuscitation after hemorrhage and sepsis has been shown to markedly reduce the development of lung injury in animals, compared with traditional resuscitation with lactated Ringer's (LR). These experiments examined the effect of HS on lung injury after hemorrhage without sepsis. The effects of HS and LR resuscitation on neutrophil trafficking, neutrophil adhesion, and neutrophil oxidative burst were studied. BALB/c mice were hemorrhaged to a mean arterial pressure of 40 torr for 1 h. Animals were resuscitated with shed blood and either 4 mL/kg of 7.5% HS or LR in twice the volume of the shed blood. Lung histology was examined 24 h after hemorrhage. Lung myeloperoxidase content and bronchoalveolar lavage fluid neutrophil counts were obtained. Peripheral blood smears were obtained to determine the neutrophil percentage. Peripheral blood neutrophil CD11b expression and neutrophil H2O2 production were assayed by flow cytometry. HS animals had less lung injury than LR animals. The mean myeloperoxidase activity in HS versus LR animals was 1.79+/-1.33 U/100 mg versus 3.0+/-1.33 U/100 mg, respectively. The percentage of neutrophils in the bronchoalveolar lavage fluid of HS animals (3.8%+/-.8) was significantly less than that of LR animals (10.8%+/-2.1). This corresponded to a significantly higher peripheral blood neutrophil count in HS animals compared with LR animals, 41% vs. 20%, respectively. There was no difference in neutrophil expression of the CD11b integrin between the HS and LR groups. The neutrophils of LR animals had basal H2O2 production that was 107% greater than that of controls; HS suppressed this hemorrhage-induced activation by > 60%. HS resuscitation after hemorrhagic shock protects against the development of lung injury. This protection is due, in part, to suppression of the hemorrhage-induced neutrophil oxidative burst. HS resuscitation offers immunomodulatory potential after hemorrhagic shock.

Risk indicators for periodontal disease in a racially diverse urban population.

A cross-sectional study of 117 subjects from a dental clinic serving a diverse population (i.e., Whites, African-Americans, Native-Americans, and Asians) was performed to evaluate risk indicators of periodontal disease. Gingival crevicular fluid (GCF) and subgingival plaque were taken at the same visit from 4 posterior sites of the most diseased sextant in each subject. Age, smoking packyears, beta-glucuronidase (beta G), neutrophil elastase (NE), myeloperoxidase (MPO), Fusobacterium nucleatum (F. nucleatum), and Porphyromonas gingivalis (P. gingivalis) were significantly (p < 0.05-0.005) correlated with attachment loss. Probing depth was significantly correlated with smoking packyears, beta G, NE, MPO, F. nucleatum and Prevotella intermedia (P. intermedia) (p < 0.05-0.005). Mean NE value of Whites was lower than the mean NE values of African-Americans, Native-Americans and Asians (p < 0.05). Whites had a lower mean beta G value compared to African Americans, and a lower mean MPO value compared to African Americans and Native Americans. The %s of patients positive for F. nucleatum, P. intermedia and Eikenella corrodens (E. corrodens) were higher in Native Americans compared to Whites. Step-wise multiple regression analysis was performed to construct models for the estimation of probing depth and attachment loss. The most parsimonious regression models which had the best R2 values included the following variables and accounted for the indicated % of variability: models 1 and 2: beta G, race, and F. nucleatum accounted for 50% of the variability in mean probing depth and 39% of the variability in a single site (first molar) for probing depth, respectively; model 3: age, beta G, and F. nucleatum accounted for 53% of the variability in mean attachment loss; model 4: age, NE, and F. nucleatum explained 35% of the variability in a single site (first molar) for attachment loss. The results suggest that age, race, smoking packyears, beta G, NE, MPO, F. nucleatum, P. gingivalis and P. intermedia are risk indicators for periodontal disease in this racially diverse urban population. Regression models which include multiple variables (i.e., demographic factors, GCF enzymes and periodontopathic bacteria) can be used to estimate periodontal disease status.

Synthetic fibronectin peptides and ischemic brain injury after transient middle cerebral artery occlusion in rats.

Leukocytes play an important role in the development of ischemia-reperfusion injury. This study was conducted to ascertain whether synthetic peptides corresponding to the cell- and heparin-binding sequences of fibronectin that disturb leukocyte adhesion molecules were effective in neuronal protection after transient focal cerebral ischemia in rats. The authors evaluated the efficacy of peptides on infarction size, leukocyte infiltration in the ischemic tissue, and neurological outcome in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. Twenty-one animals were divided into three groups: transient ischemia without treatment (Group I), transient ischemia with administration of vehicle (Group II), and transient ischemia with administration of fibronectin peptides (Group III). The mean myeloperoxidase activity (U/g wet wt) in the ischemic area was as follows: Group I, 0.19% +/- 0.05; Group II, 0.21% +/- 0.03; and Group III, 0.08% +/- 0.02. The mean size of the infarction as a percentage of the total hemispheric volume was as follows: Group I, 38.35% +/- 1.34%; Group II, 39.21% +/- 2.42%; and Group III, 25.81% +/- 4.87%. Group III showed a significant decrease in myeloperoxidase activity in the lesion and the infarction size was smaller when compared to Groups I and II (p < 0.05). The neurological grade in Group III was significantly better than in Groups I and II at 48 hours after reperfusion (p < 0.01). This study is the first to explore the therapeutic potential of synthetic fibronectin peptides in brain protection after transient focal ischemia, and the results also serve as a basis for studies of important cellular and molecular events that contribute to tissue damage.